In recent years, numerous experiments performed in a variety of model organisms have uncovered the cellular process of RNA-induced silencing. In this process, double-stranded RNA or some other form of RNA acts as a trigger for highly sequence-specific silencing of gene expression. To date, research efforts have achieved an improved understanding of the molecular mechanisms active in the RNA-induced silencing process of RNA interference (RNAi), but very little is known about the role(s) that RNAi plays in, e.g., development and disease-related processes in complex multicellular organisms.
The RNAi pathway has been robustly conserved in evolution, and initiates RNA silencing by utilizing double-stranded RNAs that are cleaved into short 21-23 nt dsRNA molecules by a cellular enzyme known as Dicer. The short dsRNA generated by Dicer cleavage are incorporated into a large multi-protein complex known as the RNA-induced silencing complex, or RISC. RISC then utilizes the short, sequence-specific RNA generated by Dicer cleavage to target and silence homologous RNA transcripts, thereby silencing the gene. As Dicer is a key enzyme in this process, a great deal of research interest in Dicer biochemistry and in the role of Dicer in this process has ensued. Dicer has been purified, and recombinant forms of this enzyme that can process dsRNA into siRNA in vitro are commercially available.
In addition to its role in generating siRNA, Dicer has also been found to generate another class of small RNA molecules referred to as microRNA (miRNA). The miRNA molecules are derived from slightly larger (˜70 nt) endogenous, non-coding RNA containing many secondary structures, including stem loops and bulges. Unlike siRNAs, the miRNAs are not perfectly complementary in sequence to their RNA targets, and those directed to mRNA targets have been demonstrated to act by complexing with the 3′ untranslated region (3′ UTR) of their target message to inhibit translation. Several experiments have suggested that these miRNAs play a role in nematode development, as mutations in miRNA induce developmental defects in nematodes. Dicer has therefore been speculated to play a developmental role in higher eukaryotes, possibly through regulation of miRNA generation.
Although a recent publication has revealed that mouse embryos homozygous for a dicer1 mutant allele die for unclear reasons prior to gastrulation (Bernstein et al. Nature Genetics 35:215-217), improved tools are required to ascertain the role of Dicer in processes such as embryonic development, tissue-specific development, disease-related processes, etc., especially in organisms of greater complexity than nematodes.